Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with median survival times of less than a year. Activating KRas mutations are present at diagnosis in up to 95% of patients, with alterations already existing in 92% of early-stage pancreatic intraepithelial neoplasms. In addition, PDAC displays rapid invasion locally, in addition to distant metastasis, processes that require multiple molecular events that are critical to target therapeutically. While the mode of invasion varies among different PDAC cells (ie, single-cell, collective), cells at the leading edge of invasion zones may develop pathfinder cell functions, which can create the microtracks through which the tumor cells will spread, by degradation of the extracellular matrix (ECM). Actin-rich membrane protrusions (invadopodia) develop in specific cell populations, which lead the migratory cohort of invading cells. Thus, it is critical to understand the mechanisms involved in the formation of these pathfinder cells and the molecular dynamics that occur at the invading edges. Among these events, mutant KRas is required for the formation of invadopodia, and specific cellular scaffolds are required for mediating the effector signaling pathways involved. For example, Ezrin, a scaffold protein that links the plasma membrane to the actin cytoskeleton, as well as other members of the ezrin-radixin-moesin (ERM) family, are present in actin-rich invadopodia and play important roles in PDAC migration. In addition, discrete pools of active RhoA at the leading and lagging edges of motile PDACs are critical for invasive behavior. We have described a novel anti-motility target (km23-1), the depletion of which was shown to diminish Ras and ERK activity, as well as TGFss1 production and cancer cell motility and invasion. It also interacts with numerous actin-regulatory proteins and can modulate RhoA activity and Ezrin expression. We hypothesize that km23-1 plays an important role in the formation and activity of invadopodia by facilitating the assembly of Ezrin- enriched complexes that further regulate Ras activity and protein kinase A (PKA)-modulated RhoA protrusion- retraction events. Therefore, in Aim 1, we will examine the ability of km23-1 depletion to inhibit PDAC invasion in organotypic models representative of the in vivo setting, which will include stromal pancreatic stellate cells. Additional studies will address the functional role of km23-1 in regulating invadopodia RhoA activity and leader cell migration. Further, since ERM and actin are required for Ras activation, we will test the hypothesis that km23-1 regulates Ezrin scaffolded complexes to control Ras activation and downstream events involved in invadopodia protrusion during PDAC invasion. In Aim 2, we will examine the effect of km23-1 depletion on tumorigenicity and metastasis in vivo to determine the potential utility of km23-1 inhibitors as anti-metastatic therapy. The results of these studies will provide significant new information regarding the spatial control of Ras and RhoA activity, as well as Ezrin/actin scaffolding, in PDAC invasion and metastasis. They will also facilitate efforts to develop novel km23-1- and Ezrin-based inhibitors for the treatment of PDAC.